The present invention is directed to the production of proteins of bacterial origin in the filamentous fungi, for example in Aspergillus and Trichoderma. Preferably, the proteins originate from actinomycetes. The compositions of the invention are useful for modifying plant biomass properties, especially to reduce the lignin content. The invention is also directed to the area of thermostable xylanases that are active at high temperatures and to a method for bleaching with the aid of the enzyme compositions of the invention.
The aim of kraft pulp bleaching is to remove the residual lignin that is left in pulp after kraft cooking. Traditionally, this has been done using chlorine-containing chemicals. Because of environmental concerns and consumer demands, alternative bleaching technologies have been desired.
The first biotechnical approach to this problem was to attack the lignin directly with lignin degrading enzymes. However, the chemistry of enzymatic lignin degradation seems to be very complicated and difficult to control.
Lignin can be degraded, if the whole microorganism that produces ligninases is used. However, treatment times are relatively long. For example, treatment times may take days, and the microorganisms need supplemental nutrients to work. It can also be difficult to control the growth of other, undesired, microbes. The use of lignin degradation by isolated ligninases or by microorganisms is the subject of much research. (see, for example, Farrell, R. L. et al., Lignocellulosics 305-315 (1992); Jurasek, L., Lignocellulosics 317-325 (1992)).
In addition to cellulose and lignin, wood pulp contains hemicellulose. Another approach is to attack hemicellulosexe2x80x94the third main component of wood. The hemicellulose in native hardwood is mainly xylan, while in softwood the hemicellulose is mainly glucomannans and some xylan. During kraft cooking, part of the xylan is dissolved into the cooking liquor. Towards the end of the cooking period when the alkali concentration decreases, part of the dissolved and modified xylan reprecipitates back onto the cellulose fiber.
In 1986, it was noticed that xylanase pretreatment of unbleached kraft pulp results in a lessened need for chemicals in the bleaching process (Viikari, L. et al., Proceedings of the 3rd Int. Conf. on Biotechnology in the Pulp Paper Ind., Stockholm (1986), pp. 67-69). Xylanase pretreatment of kraft pulp partially hydrolyses the xylan in kraft pulp. This makes the pulp structure more porous and enables more efficient removal of lignin fragments in the subsequent bleaching and extraction stages. Later, in several laboratories, the xylanase pretreatment was reported to be useful in conjunction with bleaching sequences consisting of Cl2, ClO2, H2O2, O2 and O3. See reviews in Viikari, L. et al., FEMS Microbiol. Rev. 13: (1994xe2x80x94in press); Viikari, L. et al., in: Saddler, J. N., ed., Bioconversion of Forest and Agricultural Plant Residues, C-A-B International (1993), pp. 131-182; Grant, R., Pulp and Paper Int. (September 1993), pp. 56-57; Senior and Hamilton, J. Pulp and Paper:111-114 (September 1992); Bajpai and Bajpai, Process Biochem. 27:319-325 (1992); Onysko, A., Biotech. Adv. 11:179-198 (1993); and Viikari, L. et al., J. Paper and Timber 73:384-389 (1991).
As a direct result of the better bleachability of the pulp after such a xylanase treatment, there is a reduction of the subsequent consumption of bleaching chemicals, which when chloride containing chemicals are used, leads to a reduced formation of environmentally undesired organo-chlorine compounds. Also as a direct result of the better bleachability of pulp after a xylanase treatment, it is possible to produce a product with a final brightness where such brightness would otherwise be hard to achieve (such as totally chlorine free (TCF) bleaching using peroxide). Because of the substrate specificity of the xylanase enzyme, cellulose fibers are not harmed and the strength properties of the product are well within acceptable limits.
However, it is not as simple as merely adding a xylanase treatment step. Most commercial xylanases designed for pulp bleaching are not very thermotolerant, especially when neutral or alkaline pH conditions are used. In practice, xylanases are generally inefficient or inactive at temperatures higher than 60xc2x0 C.
The cloning of xylanases has been reported from Actinomadura sp. FC7 (Ethier, J.-F. et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, R. Baltz et al., eds, (Proc. 5th ASM Conf. Gen. Mol. Biol. Indust. Microorg., Oct. 11-15, 1992, Bloomington, Ind., poster C25); bacteria (e.g. Ghangas, G. S. et al., J. Bacteriol. 171:2963-2969 (1989); Lin, L.-L., Thomson, J. A., Mol. Gen. Genet. 228:55-61 (1991); Shareck, F. et al., Gene 107:75-82 (1991); Scheirlinck, T. et al., Appl Microbiol Biotechnol. 33:534-541 (1990); Whitehead, T. R., Lee, D. A., Curr. Microbiol. 23:15-19 (1991)); and fungi (Boucher, F. et al., Nucleic Acids Res. 16:9874 (1988); Ito, K. et al., Biosci. Biotec. Biochem. 56:906-912 (1992); Maat, J. et al., in Visser, J. et al., eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp. 349-360 (1992); van den Broeck, H. et al., EP 463,706 A1 (1992), WO 93/25671 and WO 93/25693). It has been proposed by some researchers that the former genus Actinomadura should be divided into two genuses, Actinomadura and Microtetraspora, the latter including, e.g. the former A. flexuosa (Kroppenstedt et al., System. Appl. Microbiol. 13: 148-160 (1990).
It is known that Thermomonospora fusca produces thermostable and alkaline stable xylanases (EP 473,545, Sandoz). The use of hemicellulose hydrolyzing enzymes in different bleaching sequences is discussed in WO 89/08738, EP 383,999, WO 91/02791, EP 395,792, EP 386,888, EP 473,545, EP 489,104 and WO 91/05908. The use of hemicellulolytic enzymes for improved water removal from mechanical pulp is discussed in EP 262,040, EP 334,739 and EP 351,655 and DE 4,000,558. When the hydrolysis of biomass to liquid fuels or chemicals is considered, the conversion of both cellulose and hemicellulose is essential to obtain a high yield (Viikari et al., xe2x80x9cHemicellulases for Industrial Applications,xe2x80x9d In: Bioconversion of Forest and Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)). Also, in the feed industry, there is a need to use a suitable combination of enzyme activities to degrade the high xcex2-glucan and hemicellulose containing substrate.
A xylanase that is active at an alkaline pH would decrease the need to acidify the pulp prior to xylanase treatment. In addition, the temperatures of many modern kraft cooking and bleaching processes are relatively high, well above the 50xc2x0 C. that is suitable for many of the commercial bleaching enzymes. Accordingly, a need exists for thermostable xylanase preparations that are stable at alkaline pH""s for use in wood pulp bleaching processes.
The efficient and cost-effective production of thermostable xylanases is a problem, because thermostable xylanases originate mainly from relatively unstudied bacteria, which often produce only minimal or very small amounts of xylanase. Further, there may be little or no experience of cultivating these microbes in a fermentor or no fermentation processes available. Furthermore, these microbes may be unsuitable for industrial scale production. On the other hand, filamentous fungi like Aspergillus and Trichoderma are known to produce large quantities of proteins, on an industrial scale. In particular, these fungi have been shown to be suitable for production of homologous or heterologous proteins of fungal origin.
There are, however, very few reports of producing proteins of bacterial origin in filamentous fungi and as far as we know, no reports of the production of proteins of bacterial origin in Trichoderma. Accordingly, a need exists for efficient and cost-effective production of thermostable xylanases of bacterial origin. According to this invention our solution is production in the filamentous fungi and preferably in Trichoderma.
The invention is directed inter alia, to a method of expressing and secreting proteins of bacterial origin with a high level of expression and secretion. As much as 10-fold greater production and secretion levels than that previously observed have been obtained. The invention, however, may result in either lesser or greater levels of expression.
It is therefore an object of this invention to produce protein of bacterial origin in filamentous fungi such as Aspergillus or Trichoderma. Preferably, the host is Trichoderma and the proteins originate from actinomycetes.
It is further an object of this invention to provide recombinant vectors comprising a DNA sequence encoding a bacterial protein, wherein said bacterial protein encoding sequence is fused in frame with the sequence of a secreted enzyme. Preferably the bacterial protein originates from actinomycetes and the enzyme it is fused to is selected from the group consisting of homologous secretable proteins from filamentous fungi. Most preferably the enzyme is Trichoderma cellulase or hemicellulase, or at least a functional domain of said cellulase or hemicellulase. More preferably the protein from actinomycetes is a xylanase and most preferably an Actinomadura xylanase.
It is further an object of this invention to provide culture medium from the culture of hosts, into which said recombinant vectors have been transformed. Preferably the host is a Trichoderma. More preferably the host is Trichoderma reesei. 
It is further an object of this invention to provide a method of enzyme-aided bleaching comprising adding said culture medium to pulp.
It is further an object of this invention to provide a method for chemically treating plant biomass with said culture medium.